Gene mutations caused the so-called Warburg effect which showed dramatic changes in the aerobic glucose metabolic pathways in tumor cells. Specifically, glucose metabolism in tumor cells increases 200-fold over normal cells, but the lactic acid is excreted and cannot enter the mitochondrial tricarboxylic acid cycle of tumor cells. Hence glutamine becomes the necessary raw material for the production of energy in mitochondria of tumor cells, and meanwhile glutaminase is the essential enzyme assisting glutamine to be metabolized and fed into the mitochondrial tricarboxylic acid cycle. Therefore, any compound that causes the inhibition of glutamine metabolism or glutaminase activity, can inhibit the growth of tumor cells. On the other hand, elemental selenium also has an inhibitory effect on the growth of tumors, and the selenium in the human body produces different anticancer effects through a variety of metabolic mechanisms.
Ebselen (2-phenylbenzoisoselenazol-3-one) which is the best inhibitor for glutathione peroxidase (GPx), has been reported to also inhibit other enzymes, thus it is suspected to inhibit a variety of enzymes by selenium redox reactions, hence it lacks specificity and has low cellular activity.
For example, Ebselen was reported as a glutaminase inhibitor (IC50 9 nM) and a preferred inhibitor of glutathione peroxidase (GPx), but it is not potent in inhibiting glutamine dependent cancer cell growth (IC50, 20 uM).
We suspected that ebselen may not inhibit glutaminase. Therefore, we did biomolecular interaction analysis using ForteBio instrumentation, and discovered that ebselen was not good at binding glutaminase and 10 μM ebselen cannot directly combine toglutaminase. Therefore, based on the crystal structure of the allosteric site of glutaminase, we synthesized a series of new symmetric and asymmetric ebselen derivatives with the aim to make an ebselen derived KGA inhibitor. In the derivatives, the benzene ring at the 2-position of the original ebselen molecule was replaced by a straight-chain or branched alkyl substituent containing at least one carbon atom, or a long chain containing thioether, or a smaller cycloalkyl group, and the newly obtained derivatives are effective in binding to the allosteric site of glutaminase. The experiment showed that these compounds can directly bind to glutaminase, and their inhibitory activity to the growth of tumor cells (T24) increased 100-fold over ebselen. Moreover, these inhibitors that bind the allosteric site of glutaminase had good selectivity and higher potency for inhibition of tumor cell growth (<100 nM), and no damage to normal cells (10 μM). These inhibitors also had significant activity in an immunodeficient mouse model with no toxicity. These inhibitors had good inhibitory activity for various types of glutamine-dependent cancer cells (80-90%), especially urinary system tumors. The binding diagram of GAC tetramers and BPTES is shown in FIG. 1, wherein “mark 1” is the active site of substrate binding; and “mark 2” is an allosteric site for BPTES binding.
In addition, we added electro-withdrawing or electro-donating groups to the benzene ring of ebselen to increase or decrease the redox activity of compounds, optimize the pharmacological properties of compounds, the binding capacity with glutaminase, as well as the antitumor activity of compounds. The metabolism of glutamine (Gln) by glutaminase provides important material for the rapid synthesis of protein/lipid/nucleic acid in tumor cells and maintains the redox state. The growth of many tumor cells depend on glutamine, because it was fed into the mitochondrial tricarboxylic acid cycle, which makes; consequently, inhibitors of glutamine are predicted to inhibit the growth of tumors.
The invention found new drugs with high efficacy against cancer cells with low systemic toxicity. Since the growth of many tumor cells is dependent on the metabolism of glutamine in mitochondria, the inhibitors of glutaminase can inhibit the growth of tumors with little toxicity to normal cells. In the meantime, the molecular structure of ebselen was modified by replacing the benzene ring at the 2-position, with carbochains, carbocycles or derivatives thereof containing several carbon atoms, such as thioethers, or other derivatives including N, or O. These modifications are beneficial for compounds to bind with the allosteric site of glutaminase, meanwhile adding the electron-withdrawing group or electron-donating groups to the benzene ring of the selen moity can increase or decrease the redox activity of compounds, and optimize the pharmacological properties of compounds, the binding capacity with glutaminase, as well as the antitumor activity of compounds. The synthesized compounds have the ability of targeting, inhibiting mitochondrial function and blocking the energy metabolism of tumors, in addition, they showed 100 folds better inhibition of glutaminase than that of ebselen, and have a very notable competence to inhibit glutamine-dependent tumor, which can be used for the treatment of tumors of urinary system or for the post-operative cancer prophylaxis.
In additional, glutamic acid is an important neural transmitter. Overloading glutamic acid is characteristic right after the onset of ischemic stroke, therefore glutaminase inhibitor might correct the overloading and protect the injured nerve cells from death. These compounds show clear evidence of cell protection under OGD condition (oxygen glucose deficient condition) which is an in vitro mimic of hypoxia-ischemia situation.